The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Development of a Quantitative Real-time Nucleic Acid Sequence based Amplification (NASBA) Assay for Early Detection of Apple scar skin viroid

  • Heo, Seong (Apple Research Institute, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Kim, Hyun Ran (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration) ;
  • Lee, Hee Jae (Department of Plant Science, Seoul National University)
  • Received : 2018.10.07
  • Accepted : 2019.01.04
  • Published : 2019.04.01
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Abstract

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with realtime detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of $1{\times}10^4$ to $1{\times}10^{12}$ ASSVd RNA $copies/{\mu}l$ with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from $1{\times}10^7$ to $1{\times}10^{12}$ copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

Keywords

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Fig. 1. Nucleotide sequence alignments of ASSVd isolates and the position of ASSVd-specific primers and molecular beacon. Comparison of the nucleotide sequences of ASSVd isolate collected from ‘Hongro’ cultivar at the orchard of NIHHS with ASSVd-K (Korean strain, accession AF421195), A-4 (DQ362907), B-9 (DQ362906), AM1 (EU031455), A1 (HG764204), B1 (HG764201), C1 (HG764197), C2 (HG764198), C3 (HG764199), C4 (HG764200), T1 (HG764202), P1 (HG764203), N1 (HG764205). ASSVd isolate which identified in this study had 100% sequence homology with ASSVd-K. Identical nucleic acid residues are shown in black, whereas dashes indicate gaps in the nucleotide sequences introduced to optimize alignment. The primers and molecular beacons sequences for ASSVd are indicated by red rectangles.

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Fig. 2. A dendrogram which constructed from the sequences of ASSVd isolates; accessions K (AF421195), B-9 (DQ362906), A - 4 ( D Q 3 6 2 9 0 7 ), A M 1 ( E U 0 3 1 4 5 5 ), A M 2 ( E U 0 3 1 4 5 6 ), P P 1 ( E U 0 3 1 4 6 7 ), PE1 (EU031477), AP1 (EU031487), C1 (HG764197), C2 (HG764198), C3 (HG764199), C4 (HG764200), B1 (HG764201), T1 (HG764202), P1 (HG764203), A1 (HG764204), N1 (HG764205), ap (HQ840722), and ADFVd (NC_003463), CBLVd (M74065), and PBCVd (NC_001830) belong to the genus Apscaviroid. It was constructed by the maxium likelihood method based on Tamura-Nei model with 1,000 bootstraps.

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Fig. 4. The standard curve of ASSVd RNA using real-time NASBA. (A) Real-time NASBA amplification plot based on TTP value versus fluorescence. (B) Relationship of TTP to ASSVd RNA copy number.

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Fig. 5. Comparison of sensitivity to detect ASSVd RNA molecules by RT-PCR and real-time NASBA. (A) End-point detection of ASSVd by RT-PCR. (B) Relative fluorescence with a dilution series by real-time NASBA.

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Fig. 3. Sensitivity of ASSVd real-time detection using molecular beacon to determine the optimization of the assay. Optimization of (A) the primers, (B) KCl and (C) ITP concentration with the highest fluorescent signal or the lowest time to positive (TTP) value.

Table 1. Sequences of the primers and molecular beacon used in RT-PCR and real-time NASBA assays for detecting ASSVd in apple trees

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